Middle ear infection or otitis media (OM) is a highly prevalent pediatric disease worldwide. There were nearly twenty-five million physician's office visits made for OM in 1990, and available evidence suggests that the incidence is increasing. While only very rarely associated with mortality, the morbidity associated with OM is significant. The socioeconomic impact of OM is also great. Direct and indirect costs of diagnosing and managing OM exceed $5 billion annually in the U.S. alone. Clearly, there is a tremendous need to develop more effective and accepted approaches to the management and preferably, the prevention of OM. Vaccine development holds the greatest promise and would be the most cost-effective method to accomplish this goal. However, progress in terms of vaccine development for nontypeable Haemophilus influenzae (NTHI), the Gram-negative pathogen that both predominates in chronic otitis media with effusion or OME, as well as being a significant etiologic agent of acute OM, continues to be hampered by our incomplete understanding of the pathogenesis and immunobiology ofOM, a polymicrobial disease caused by one or more of the three predominant bacterial pathogens, whose ability to invade the tympanum is facilitated by virtually any upper respiratory tract (URT) virus. During the past 4 years, we have: sequenced and will soon complete the annotation of the genome of an OM isolate of nontypeable Haemophilus influenzae (NTHI); developed and used DNA plasmid based microarrays to conduct strain comparison studies for two clinical OM isolates; developed a promoter trap system in NTHI with which we have monitored gene expression in vivo, during experimental OM; and have developed a signature tag mutagenesis system in NTHI that provided us with a complementary system to identify genes that are essential for colonization and induction of OM. Not only are all these tremendous resources available to us to capitalize upon as we extend our studies of the pathogenesis of NTHI-induced OM, but they have also already resulted in the identification of multiple potential new virulence determinants for NTHI. We propose experiments for the next funding period designed to continue to enhance our understanding of both NTHI pathogenesis in OM at the molecular level as well as further our investigation of a focused group of novel virulence determinants. We will first rigorously assess the feasibility of using lux-expressing NTHI strain 86-028NP to provide a non-invasive, whole animal imaging system to be applied to, and significantly advance, our studies of both pathogenesis and vaccine-mediated prevention of OM. Secondly, we will use a chinchilla super infection model to assess the protective efficacy of one well-developed candidate antigen and one novel and highly promising, but less developed candidate, for ability to prevent ascending OM after intranasal immunization. We will also continue to identify and characterize putative virulence determinants and assess their potential as vaccine or therapeutic targets via a variety of methodologies, including gene expression profiling by microarray analysis. Overall, our studies will lead to an improved understanding of OM caused by NTHI and lead to new strategies to prevent OM.